MITOCHONDRIAL DYSFUNCTION is an important component of many of the pathologies associated with aging, such as type 2 diabetes mellitus, Alzheimer's disease, Parkinson's disease, and some cancers. Indeed, mitochondria have been implicated overall in the aging process, although the mechanisms are not fully understood. One of the most widely accepted theories of aging, the oxidative stress theory, suggests that the aging process involves the accumulation of oxidative damage to mitochondria and other cellular components. Oxidative damage is induced by reactive oxygen species (ROS), produced primarily as a by-product of mitochondrial oxidative phosphorylation. As mitochondrial ROS can cause damage to mitochondrial DNA, proteins, and membrane lipids, a self-perpetuating and destructive cycle can ensue in which increased ROS production leads to incremental damage and further ROS production. Calorie restriction (CR), without malnutrition, is a well-known dietary intervention that consistently increases life span by delaying the aging process in a wide variety of animal species. The mechanisms underlying aging retardation by CR are poorly understood. However, it has been suggested that they may involve a decrease in cellular oxygen consumption and ROS production. Based on solid preliminary data in both pre-clinical rodent models and in humans participating in a 6 month study of CR (phase 1 of this U01), we propose the hypotheses that 2 years of 25% CR will increase mitochondrial biogenesis and increase coupling in vivo; that these changes will be associated with a reduction in ex vivo mitochondrial ROS production and accumulated mitochondrial ROS damage. To test this hypothesis, we will conduct an ancillary study to the multi-center CALERIE phase 2 U01 at the Pennington Biomedical Research Center. This multi-disciplinary project will use newly developed state-of-the- art in vivo measurement of skeletal mitochondrial capacity / resting ATP synthesis [31P MR spectroscopy] combined with in vivo measurement of O2 uptake [deconvolution of high resolution optical spectrometry] to measure mitochondrial coupling during a two year 25% CR. These measures will be paired with measures of mitochondrial enzyme content and mitochondrial ROS production assays performed on fresh skeletal muscle biopsies. Importantly, we will measure mitochondrial ROS production and ROS damage to fully test the hypotheses at hand; namely that CR will increase coupling, reduce ROS production and decrease ROS damage. These totally novel studies of mitochondrial adaptations to caloric restriction will directly test the first and critical aspects of the oxidative stress theory of aging in the longest controlled study of CR in humans planned to date. PUBLIC HEALTH RELEVANCE: The energy producing cellular organelles called mitochondria produce free radicals producing oxidative stress. Over time, it is thought that this oxidative stress damages mitochondria as a major part of aging. This protocol aims to test the hypothesis that reducing energy intake in non-overweight people will make mitochondria more efficient at producing ATP, thereby reducing oxidative stress and accumulated oxidative damage.